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Anaplasma marginale causes mild to severe hemoparasitic disease resulting in significant morbidity and mortality in cattle worldwide. In the absence of universally efficacious vaccines, antimicrobial therapy combined with biocontainment and biosecurity strategies are critical to control anaplasmosis. Herein, we compared the effect of oxytetracycline, imidocarb and enrofloxacin on A. marginale isolates in short-term erythrocyte cultures. Electron micrographs detailing antimicrobial-induced changes in rickettsial morphology were scored (0-4) based on ultrastructural changes. These were compared to fluorochromatic changes detected by flow cytometry (FACS) using conversion of hydroethidine (HE) to ethidium bromide (EB) by living organisms to assess viability. A. marginale infectivity in selected cultures was confirmed by subinoculation into susceptible calves. Morphology scores were analyzed using Chi-squared tests and compared to FACS data by ANOVA with isolate, drug and concentration as co-variates in the model. Only the Virginia and Oklahoma isolates exposed to 1.0 microg /ml imidocarb and the Oklahoma isolate exposed to 4.0 microg /ml enrofloxacin were sterilized following antimicrobial exposure. Rickettsia with morphology scores of 0 had significantly more EB positive cells than inclusions with morphology scores of 4 (p=0.039). There was also a significant association between ultrastructural changes and infectivity (p=0.0047). Furthermore, the percent EB positive cells in the antimicrobial exposed cultures was highly predictive of the probability of infectivity (p=0.0026). This is the first study describing ultrastructural changes in A. marginale following exposure to enrofloxacin and imidocarb. These findings demonstrate that FACS and electron microscopy are useful tools to screen new antimicrobials for the use in anaplasmosis chemotherapy.

Previous studies have shown that one Brazilian Anaplasma marginale isolate presents an inclusion appendage (tail), while other isolates do not present such inclusion. Studies on tick transmission have been carried out with tailless isolates but little is known about transmission of tailed isolates by Boophilus microplus. Two splenectomized calves were experimentally inoculated with the tailed A. marginale isolate. During ascending rickettsemia, B. microplus larvae, free from hemoparasites, were fed on the calves and the resulting nymphs, adult males and engorged females were examined by optic and electronic microscopy. No A. marginale colonies were observed in the gut cells of engorged females and the larvae originated from them did not transmit A. marginale to susceptible calves. In addition, no colonies of A. marginale were seen in the gut cells or in salivary glands of adult males and nymphs. These results suggest that B. microplus is not the biological vector for this tailed isolate.

Colonies of Anaplasma marginale Theiler were studied in midgut epithelial cells of nymphal Dermacentor andersoni Stiles that had become infected by feeding on splenectomized calves with anaplasmosis. Colonies of A marginale were not observed in nymphal ticks killed during the 6-day feeding period, but were present in sections of midgut epithelial cells of ticks killed as early as 5 days after repletion. Colonies of A marginale also were present in ticks examined throughout development to the adult stage. Two distinct morphologic types of colonies were observed and categorized by light microscopy as nymphal types 1 and 2. Colonies that were morphologically indistinct with characteristics common in both types were termed transitional nymphal (TsN) colonies. Nymphal type 1 (Ny1) colonies were observed at 5 days after repletion and nymphal type 2 (Ny2) colonies were first observed at 20 days after feeding. Representatives of each colony type were selected by light microscopy and were sectioned for electron microscopy. The Ny1 contained small particles and large, round reticulated forms, some of which appeared to be dividing by binary fission. The Ny2 also contained reticulated organisms, but they were rod-like in shape, and there was no morphologic evidence of binary fission. Organisms within Ny2 appeared to be surrounded by 2 double-layered membranes. Electron-dense forms, commonly observed in adult ticks, were not seen in colonies (Ny1, Ny2, or TsN) from nymphal ticks.